The most common rationale for the fluorescent measure is the need for sensitivity, or a high signal-to-noise level afforded by a dark-field measure of fluorescence. The main object of the fluorescent measure is to admit only the fluorescent signal (range of wavelengths) to a sensor, and to reject excitation wavelengths. Fluorescent measurement sensitivity is usually limited by the noise associated with a background of stray light that compromises the dark-field. Background stray light may emanate from a sample or the measurement system; stray light from the system is mitigated by spectral filtration. Spectral filtration of light is essential to fluorescent measurement, wherein stray light from excitation energy must be eliminated from an optical light path that directs the essential fluorescent energy signal to a photo sensor.
For many fluorescent applications, the greatest sensitivity is obtained by exciting with a wavelength of light that is only tens of nanometers below the emission wavelength, where the excitation/emission difference is called the Stokes Shift of the fluorochrome. Dichroic (interference) filters are commonly applied, since they can be designed and manufactured to enable the appropriate rejection of stray excitation light from productive emission light. While high-pass filters that absorb light with a chromaphore can be designed and manufactured to absorb excitation and pass emission, they generally do not enable productive rejection and transparency over the demanded tens of nanometers and absorbing chromaphores have a tendency to fluoresce (contribute noise in the domain measurement wavelengths). Further, the design/manufacture of a dichroic filter is amenable to enabling a bandpass of transmitted light, designated by a cut-on and cut-off wavelength of an emission spectrum (according to the conventional spectrum of increasing wavelength), providing a transmission window for the emission. The well-designed bandpass dichroic is essential to the sensitive fluorescent measure, since materials other than the fluorochrome targeted for measure may fluoresce. To the likely extent that those other materials fluoresce out of the bandpass designed for a targeted fluorochrome, their emissions are rejected the sensitivity of the fluorescent measure improves.
An increasingly common need for the fluorescent measure is the imaging applications, wherein a combination of lenses direct the fluorescent energy signal to an area sensor such as film or an electronic sensor (CCD array). To further the sensitivity and dynamic range of the fluorescent measurement, it is increasingly common to use a cooled CCD array sensor. To further the speed and acuity of the imaging, and to broaden the operating range of the optics, very sophisticated lenses (many elements) must be used.
A difficulty in applying an appropriately designed dichroic filter to the imaging application is that the dichroic filter bandpass is a function of the angle of incidence of light on the filter plane, as shown in FIG. 1A. Specifically, light incident at off-normal incidence traverses a longer optical distance in the interference coating; hence the cut-on (and cut-off) wavelength of the dichroic filter decrease, or the bandpass blue-shifts. Hence, for the wide-angle of view enabled by a sophisticated lens, the emission filter bandpass presents a blue-shift that increases with image radius (FIG. 1B). The radial-blue shift can cause a severe artifact in a high-sensitivity fluorescent imaging application if the blue-shifted cut-on of the emission filter significantly overlaps the cut-off of the excitation light. Consequent stray excitation light infiltrates the periphery of the field of view, producing imaging artifacts, often viewed as bright rings in the otherwise dark field. Generally, the bright ring artifacts are a reflection of the circular features in the multi-element lens that reflect the stray light back to the dichroic filter, which rejects the light back into the lens and ultimately to the sensor. An example of the imaging artifact is shown in FIG. 2A.
One method of managing blue-shifted bandpass in a wide-angle imaging system is the placement of a dichroic emitter filter behind the lens elements (shown in FIG. 1A as element—in dashed lines 100). The method is somewhat more effective at reducing blue shift, since the wide angle rays can be more suppressed in the well-designed lens. Although effective, the method fails to reject excessive excitation light from multiple lens elements having reflective and fluorescent materials and surfaces within the lens. A resulting image artifact appears as a haze (not necessarily uniform) in the dark field, which contributes to the background noise.
There is thus a need for a cost effective technique that suppresses imaging artifacts that impede sensitive fluorescent measures.